PROJECT SUMMARY Programmed cell death 1 (PD-1) is one of the key co-inhibitory molecules upregulated upon T cell activation and is a hallmark of T-cell exhaustion. Despite the fact that PD-1 blockade has become a revolutionary strategy in treating cancer and infectious diseases, the mechanobiology of PD-1 has not been studied. This is the gap that tne present Physical Science ? Oncology Project (PS-OP) aims to fill. The significance of the PS-OP?s unique mechanobiology angle lies in the therapeutic potential of targeting the mechanoregulation of PD-1 to treat a wide variety of diseases, including melanoma, which is the focus of this PS-OP. The approaches of the PS-OP?s multidisciplinary team combine four physical science (PS) tools with two mouse models of melanoma. They represent the unique strength of the Zhu lab and the Ahmed lab, and enable investigation of PD-1 mechanobiology in silico, in vitro and in vivo. The first PS tool is DNA-based mechanical tension probes (MTP) and tension gauge tethers (TGT) that report and limit, respectively, cell-generated forces on PD-1. The second PS tool is three biomembrane force probe (BFP)-based single-molecule methods that quantify force regulation of in situ PD-1?PD-ligand interactions with concurrent imaging of intracellular signals in single cells. The third PS tool is molecular dynamics (MD) simulations that reveal structural changes of PD-1 in complex with its ligands under force and the bonding dynamics at atomic level. The fourth PS-tool is microfluidic-based devices for cell trapping, stimulation, and analysis. Preliminary studies of the project demonstrate that: 1) cells actively pull on PD-1; 2) force on PD-1 elicits catch bonds to regulate ligand bonding; 3) force induces rearrangement of the PD- 1?PD-L2 binding interface to form new atomic-level interactions; 4) mutating specific amino acids on PD-1 alters its force, catch bond and function; and 5) PD-1?s inhibitory signal suppresses antigen recognition by disrupting the synergy between TCR and CD8 in pMHC binding. These data support the hypothesis that force critically regulates ligand bonding and signaling of PD-1; as such, targeting the PD-1 mechanoregulation may represent a novel approach to immunotherapy. This hypothesis will be tested by three specific aims: 1) Determine the forces on PD-1 and their impact on PD-1 ligand bonding, signaling and function; 2) Modulate T cell function by targeting PD-1 mechanoregulation; and 3) Investigate the therapeutic potential of manipulating PD-1 catch bonds in tumor mouse models. These studies will elucidate the mechanisim of PD-1 signaling, improve one?s understanding of CD8+ T-cell responses to melanoma, and suggest new immunotheraputic strategies to treating cancer.